Polynucleotide separation method and apparatus therefor

ABSTRACT

Different probes each having a specific base sequence are immobilized to each of independent areas formed on the surface of a substrate, complementary polynucleotides in a sample solution are hybridized to the probes, and each of the independent areas on the substrate is heated and then cooled in sequence, and hence the solution is recovered to extract different polynucleotides separately corresponding to individual probes.

This is a continuation application of U.S. Ser. No. 09/666,883, filedSep. 20, 2000, (now U.S. Pat. No. 6,361,953) which is a continuationapplication of U.S. Ser. No. 09/522,465) filed on Mar. 9, 2000 (now U.S.Pat. No. 6,218,126), which is a continuation application of U.S. Ser.No. 09/329,318, filed Jun. 10, 1999 (now U.S. Pat. No. 6,093,370). Thisapplication is related to U.S. Ser. No. 09/790,872, filed on Feb. 23,2001.

BACKGROUND OF THE INVENTION

The present invention relates to a method for selectively extracting atarget polynucleotide having a specific base sequence from apolynucleotide mixture sample having a plurality of different sequencesor from cells, and an apparatus therefor.

A DNA chip is one of means for detecting a specific base sequence of DNAquickly and easily. It utilizes micropatterning techniques used inmicroprocessing of semiconductors and the complementarity of DNA. Inthis technique, DNA sequence of a sample solution is analyzed in thefollowing manner: Single stranded-oligonucleotides as probes havingdifferent sequences each of a length of approximately 8-9 bp areimmobilized separately onto each of two-dimensionally split individualareas on a substrate; the sample solution containing DNA is addeddropwise to the substrate to hybridize the DNA separately with each ofprobes in each of the areas of the substrate while attaching afluorescent dye to the hybrids simultaneously in the hybridization step;and the magnitude of the hybridization between the probes and DNA in thesample solution is optically determined through emitted fluorescence toanalyze the DNA sequence in the sample solution.

U.S. Pat. No. 4,446,237 discloses a method for capturing a targetoligonucleotide (DNA or RNA) as a probe on a solid phase. According tothis method, the oligonucleotide in a sample solution is denatured intosingle strands by heating, which is then immobilized on the surface of anitrocellulose membrane. S. R. Rasmussen et al. describe another methodfor capturing a target oligonucleotide sample on a solid phase inAnalytical Biochemistry 198, 128-142(1991). According to the method, thephosphate group at the 5′-end is activated by using 1-methylimidazoleand 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The activatedpolynucleotide is then immobilized onto a polystyrene microplate havinga secondary amine on its surface. The activated 5′-end phosphate groupreacts with the secondary amine, so that the 5′-end of thepolynucleotide is covalently immobilized onto the microplate surface.

As thus described, a target polynucleotide in a sample solution can becaptured and analyzed by selectively hybridizing a target polynucleotide(DNA or RNA) with a complementary oligonucleotide immobilized as a probeon a membrane surface. The DNA chip is on the basis of this concept.

Okano et al. describe a method for extracting target polynucleotides byhybridizing the target polynucleotides to probes immobilized on a chip,heating the chip to denature the captured polynucleotides on the probesto separate and collect them from the chip in U.S. Pat. No. 5,607, 646.

A method for selectively extracting a target polynucleotide by utilizingdifference in rates in electrophoresis of polynucleotides in gel hasbeen in wide use.

SUMMARY OF THE INVENTION

The gene analysis technology using a DNA chip described in the above isa technique for hybridizing and analyzing a target polynucleotide (DNAor RNA) by complementarily hybridizing single-stranded polynucleotidesderived from the target polynucleotide in a sample solution with a probe(a specific single stranded-oligonucleotide) with a length of 8-9 bpformed on a substrate. This technique is never directed to furtherextract the captured or hybridized DNA or RNA singlestranded-polynucleotide on the probe from the substrate selectively.

Conventional techniques give no consideration of extracting a targetpolynucleotide to be extracted directly from cells.

According to conventional separating techniques using electrophoresis,the mobility of each of polynucleotides is relative to each other andfluctuates with changes of electrophoresis conditions, and thusidentification of an extracted sample solution component is required. Inaddition, exact separation and purification of a trace quantity of atarget polynucleotide is difficult because diffusion in theelectrophoresis step can invite contamination of polynucleotides witheach other.

It is, therefore, an object of the present invention to provide aprocess and apparatus for selectively extracting a trace quantity of atarget polynucleotide (DNA or RNA) having a specific base sequencerapidly with a high precision.

The invention proposes, to achieve the above object, selectiveextraction of a target polynucleotide alone from a sample solution bymodifying each of independent split areas on the surface of a substrateseparately with each of probes (specific oligonucleotides) havingdifferent base sequences respectively, hybridizing polynucleotides (DNAor RNA) in the sample solution separately to the probes, and thenheating a specific area alone of the substrate selectively to allow apolynucleotide alone complementarily hybridized with the heated probe toliberate from the probe.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, objects, and advantages of the presentinvention will become apparent upon a consideration of the followingdescription of the invention when read in conjunction with the drawings,in which:

FIG. 1 is a gene processing flowchart for demonstrating where the methodand apparatus for separating polynucleotides according to the inventionare located in analysis and selective extraction process of gene;

FIG. 2 is a schematic diagram illustrating a basic configurationaccording to a first embodiment of the invention;

FIG. 3 is a schematic diagram illustrating a first means for heating aspecific area on a substrate of the first embodiment;

FIG. 4 is a schematic diagram illustrating a second means for heating aspecific area on the substrate according to the first embodiment;

FIG. 5 is a schematic diagram illustrating a third means for heating aspecific area on the substrate according to the first embodiment;

FIG. 6 is a schematic diagram illustrating a practical configuration ofa polynucleotide separation cell according to the first embodiment;

FIG. 7 is a sectional view along with the lines A—A of the cellillustrated in FIG. 6;

FIG. 8 is a sectional view along with the lines B—B of the cellillustrated in FIG. 6;

FIGS. 9A, 9B and 9C are timetables respectively illustrating thenucleotide separation process according to the first embodiment of theinvention;

FIG. 10 is a diagram illustrating the relationship between a substrate 1having a target polynucleotide hybridization area and an optical systemfor detecting the hybridization in the target polynucleotidehybridization area, according to a second embodiment of the invention;

FIG. 11 is a sectional view along with the lines A—A of the substrate 1shown in FIG. 10;

FIGS. 12A, 12B and 12C are sectional views along with the lines B-B′,C—C and D—D, respectively, of a temperature control unit 133 of thesubstrate 1 according to the second embodiment.

FIG. 13 is a block diagram illustrating a polynucleotide separation cell251 and related devices thereto according to the second embodiment;

FIG. 14 is a sectional view along with the lines G—G of thepolynucleotide separation cell 251 according to the second embodiment;

FIG. 15 is a timetable demonstrating the separation process in thepolynucleotide separation cell according to the second embodiment;

FIG. 16 is a diagram illustrating stable immobilization of probes on asubstrate 1 according to a third embodiment of the invention;

FIGS. 17A, 17B and 17C are diagrams respectively illustrating testresults for verifying the advantages of the substrate according to thethird embodiment;

FIGS. 18A and 18B are diagrams illustrating efficient extraction of atarget oligonucleotide hybridized to a probe on the substrate accordingto the third embodiment;

FIG. 19 is a diagram schematically illustrating results ofelectrophoresis, demonstrating the fractionation of a doublestranded-polynucleotide according to the third embodiment;

FIG. 20 is an external view of a capillary applicable to a fourthembodiment of the invention;

FIG. 21 is a sectional view along with the lines A—A of the capillaryapplicable to the fourth embodiment;

FIG. 22 is a diagram of an illustrative configuration for immobilizing aprobe to a target polynucleotide hybridization area;

FIG. 23 is a general view of the configuration of a polynucleotideseparation apparatus according to the fourth embodiment;

FIG. 24 is a sectional view along with the lines B—B of a polynucleotideseparation module 431 of the fourth embodiment;

FIG. 25 is a diagram illustrating a variation of the polynucleotideseparation module 431 according to the fourth embodiment shown in FIG.24;

FIG. 26 is a diagram illustrating another variation of thepolynucleotide separation module 431 according to the embodiment shownin FIG. 24;

FIG. 27 is a diagram illustrating a variation of the polynucleotideseparation module 431 according to the embodiment shown in FIG. 26;

FIG. 28 is a diagram illustrating basic elements of a fifth embodimentof the invention;

FIG. 29 is a diagram illustrating a basic configuration of the fifthembodiment including a combination of a polynucleotide separation cellhaving the basic elements illustrated in FIG. 28 and an optical system;

FIG. 30 is a sectional view along with the lines A—A of the separationcell illustrated in FIG. 29;

FIG. 31A is a diagram illustrating the state where target polynucleotidehybridization areas are formed on the surface of the substrateillustrated in FIG. 28;

FIG. 31B is a diagram illustrating the state where a blood sample isintroduced into the polynucleotide separation cell and white blood cellsfloat over the surface of the substrate;

FIG. 31C is a diagram illustrating the state where white blood cells areattracted to the target polynucleotide hybridization areas by analternating field and placed separately on each of areas;

FIG. 31D is a diagram illustrating the state where white blood cellscapable of making antibody response to antigen substances alone arelabeled and become to emit fluorescence;

FIG. 31E is a diagram illustrating the state where only one white bloodcell of those hybridized on the individual target polynucleotidehybridization areas is destroyed and disappears from the substrate;

FIG. 32A is a diagram illustrating the state where probes areimmobilized on one target polynucleotide hybridization area;

FIG. 32B is a diagram illustrating the state where a white blood cell isinducted and hybridized to the target polynucleotide hybridization area;

FIG. 32C is a diagram schematically illustrating the state wherepolynucleotides and proteins migrate by electrophoresis respectively inthe opposite direction to each other; and

FIG. 32D is a diagram schematically illustrating the state wherepolynucleotide not hybridized to probes of the target polynucleotidehybridization area migrate by electrophoresis.

DESCRIPTION OF THE PREFERRED EMBODIMENT

FIG. 1 is a gene processing flowchart for demonstrating where the methodand apparatus for separating polynucleotides according to the inventionare located in analysis and selective extraction process of gene.Processes 801 and 802 are preparative processes respectively for samplecell introduction and sample solution introduction, respectively. Thepresent invention can be applied both to cells and solutions as samples.Process 803 is a cell component extraction process. Process 804 is a PCRamplification process, being conducted when a target polynucleotide hasa low concentration. Process 805 is a process for sample DNA selectionon a plurality of independent areas, which characterizes the invention.Process 806 is a selected sample observation analysis process. Process807 is sample DNA extraction process from the independent areas.

According to the invention which includes a process for sample DNAselection on a plurality of independent areas and wherein each DNA isextracted from each of the areas, efficient fractionation can beachieved, as described in detail hereinbelow.

Embodiment I

FIG. 2 is a schematic diagram illustrating a basic configuration of afirst embodiment of the invention. Substrate 1 is for hybridizing andselecting a target polynucleotide to be extracted on its surface,comprising substrate base 13 and target polynucleotide hybridizationareas 14 which are two-dimensionally mounted on the surface of thesubstrate base 13 by splitting to the order of 1 micrometer square bymeans of a light exposing technique. A plurality of probes (singlestranded-oligonucleotides) having a different sequence for each areawith a length of about 8-9 bp are individually immobilized on thesurface of each of the target polynucleotide hybridization areas 14. Theprobes are oligonucleotides complementary to a target polynucleotide ina sample solution. When the sample solution reaches the surfaces of thetarget polynucleotide hybridization areas 14, the probes hybridize withthe target polynucleotide. In other words, the target polynucleotide iscaptured by the probes. Upon the hybridization between the probes andthe target polynucleotide, a fluorescent dye simultaneously attaches tothe hybrids. Heater 31 is provided on the back of the substrate 1 andcapable of heating the whole surface of the substrate 1 from around roomtemperature up to about 95° C. The substrate 1 is disposed in thedirection perpendicular to the optical axis of object lens 15. Each oflight source 16 for fluorescence observation and infrared laser source17 for convergent light beam irradiation is disposed in the directionperpendicular to the optical axis of the object lens 15. Dichroicmirrors 181, 182 are disposed at the positions where the optical axis ofthe object lens 15 intercepts with the optical axes of the light sources16 and 17 respectively, to induce lights irradiated from the lightsources 16 and 17 respectively to the objective lens 15. The light fromthe light source 16, the fluorescence from the florescent dye and thelight from the light source 17 may preferably have a differentwavelength from each other. The light from the light source 16 can beconverged through bandpass filter 151 to adequate wavelengths forexciting the fluorescent dye. The exciting light induced to theobjective lens 15 is converged to excite the fluorescent dye attached tothe target polynucleotide hybridized and selected on the substrate. Theexcited fluorescent dye emits fluorescence in an intensity proportionalto each amount of the target polynucleotide hybridized to probes of eachof the individual areas of the substrate. Mirror 183 is to reflect alight beam which is obtained by collecting the emitted fluorescencethrough the objective lens 15. Emission filter 152 is to selectivelytransmit only a light beam having a wavelength range of fluorescence tobe detected. Detector 191 is to detect the intensity of the fluorescencetransmitted through the emission filter 152.

The spatial distribution of the amount of the target polynucleotide inthe sample solution which has been hybridized to the probe immobilizedon the substrate can be obtained as a fluorescence image by movingmutually the objective lens 15 and the substrate 1 and scanning thesurface of the substrate 1 in sequence with the exciting light inducedthrough the objective lens 15 to find the spatial distribution of thefluorescence emission intensity emitted by the fluorescent dye. When thefluorescent dye attached with the target polynucleotide is excited forobservation, the use of an exciting light having a wavelength in thevisible region and a fluorescent dye capable of fluorescing by means ofa visible light are preferred in the observation of the fluorescencefrom the dye attached with the target polynucleotide. An autoemissionfluorescence of the target polynucleotide can also be observed insteadof light emission of the fluorescent dye attached to the targetpolynucleotide. In this case the exciting light preferably has awavelength of near UV region ranging from 280 nm to 400 nm.

The fluorescence emission intensity of the fluorescent dye decreases inproportion to an increasing temperature through the function of thethermal quenching effect. By using this phenomena, an area specifictemperature increase in the target polynucleotide hybridization areas onthe substrate can be determined according to the present embodiment.According to the apparatus illustrated in FIG. 2, changes of thefluorescence emission intensity of the fluorescent dye attached with thetarget polynucleotide, which has been detected by the detector 191 canbe analyzed with analyzer 192 based upon the temperature-dependency ofchanges of the fluorescence emission intensity, and the temperature ofthe target polynucleotide hybridization areas can thus be estimated.

An infrared light emitted by the infrared laser source 17 is reflectedon the dichroic mirror 182 downward, converged on the objective lens 15and irradiated to a target polynucleotide hybridization area 14 formedon the substrate 1. In the target polynucleotide hybridization area 14,a thin film layer or particle layer formed on the substrate 1 absorbsinfrared light to evolve heat and thereby to area-specifically increasethe temperature in the sample solution. The convergent position of thelight from the laser source 17 converged on the objective lens 15 on thesurface of the substrate 1 can be changed arbitrarily by moving theobjective lens 15 or moving the substrate 1. To avoid damage to thehybridized target polynucleotide, the use of a light having a wavelengthregion not to be absorbed by the target polynucleotides is preferred. Byway of illustration, near infrared light having a wavelength of equal toor more than 800 nm or visible light having a wavelength of equal to ormore than 400 nm is preferably used.

It is known that a single stranded-polynucleotide such as DNA or RNA,being complementary to another single stranded-polynucleotide such as aprobe, forms base pairs through hydrogen bonds with the complementarypolynucleotide to form a double stranded-polynucleotide; and that thedouble stranded-polynucleotide formed by base pairs through hydrogenbonds denatures to form single stranded-polynucleotides when thetemperature of a sample solution is increased up to about 95° C.Accordingly, by irradiating the convergent infrared ray to a specifictarget polynucleotide hybridization area 14 on the substrate to increaseits temperature up to 95° C., the target polynucleotide hybridized tothe probe on the specific target polynucleotide hybridization area canselectively separated.

The probes on the substrate 1 and polynucleotides in the sample solutionabove the substrate 1 can be elongated when a planer electrode is placedon the substrate 1 and an alternating field is applied between theplaner electrode and the substrate 1. Consequently, nonspecifichybridization between the probes and polynucleotides in the samplesolution can be reduced and denaturation of the pairs between the probesand the target polynucleotide through the convergent light canefficiently be achieved.

The denaturing temperature of the target polynucleotides hybridized tothe probes increases in proportion to the amount of hydrogen bondstherebetween, and polynucleotides can selectively be fractionated inaccordance with the magnitude of hybridization matching between thetarget polynucleotides and the probes by controlling the denaturingtemperature. In other words, the target polynucleotides hybridized tothe probes begin to denature with an gradually increasing temperature,whereas the less the polynucleotides have hydrogen bonds, the earlierthey begin to denature. The target polynucleotides can, therefore, beselectively fractionated in consideration with the magnitude of specificbonds by feedback-controlling the output of the laser source 17 basedupon the temperature information obtained through the analyzer 192 andadjusting the temperature of the target polynucleotide hybridizationareas 14.

The variations of the denaturing temperatures of target polynucleotideshybridized to probes can be minimized by adding a tertiary ammonium saltsuch as tetramethylammonium chloride to the sample solution.

FIG. 3 illustrates a first means for heating a specific area of thetarget polynucleotide hybridization areas 14 on the substrate. Accordingto the present embodiment, substrate base 13 is composed of electricallyconductive film 131 provided on the surface side and thermallyconductive insulating substrate 132. Onto the electrically conductivefilm 131 are placed a plurality of target polynucleotide hybridizationareas 141, 142, 143, 144, 145 and 146. Each of the target polynucleotidehybridization areas has a dual-layer structure composed ofphotoabsorbing layer 21 such as an aluminium oxide thin film andheat-insulating layer 22. Probes (oligonucleotides) 41, 42, 43, 44, 45and 46 each having a length of 8-9 bp are respectively immobilized tothe surface of each of the target polynucleotide hybridization areasseparately. FIG. 3 schematically illustrates the state where targetpolynucleotides 41′, 42′, 43′ and 46′ are respectively hybridized onlyto the probes 41, 42, 43 and 46 of these probes. By identifying thetarget polynucleotide hybridization areas through fluorescenceobservation, the complementarity of the target polynucleotideshybridized to the probes among polynucleotides in the sample solutioncan be estimated. In the embodiment illustrated in the figure, theprobes in the areas 141, 142, 143 and 146 complementarily hybridize tothe target polynucleotides, whereas those in the areas 144 and 145 donot, indicating that there is no polynucleotide having complementaryrelation with the probes in the areas 144 and 145.

Convergent light 51 is then irradiated through the objective lens 15 tothe target polynucleotide hybridization area 142 where the targetpolynucleotide 42′ is hybridized to the probe 42; the photoabsorbinglayer 21 in the area 142 absorbs the convergent light 51 and evolvesheat. The heat from the photoabsorbing layer 21 in the area 142 allowsthe vicinity of the area 142 to increase its temperature up to about 95°C., and hence hydrogen bonds between the probe 42 and the targetpolynucleotide 42′ are dissociated to denature the target polynucleotide42′ along which has been hybridized to the area 142. When the size of anarea where the convergent light is converged is smaller than that of aunit target polynucleotide hybridization area, the light axis should beadjusted to ensure that the convergent area is within the targetpolynucleotide hybridization area. When a unit target polynucleotidehybridization area has a smaller size than the convergent area of theconvergent light, individual areas should preferably be arranged in sucha manner that gaps between individual target polynucleotidehybridization areas are sufficient and only one area is to be heated bythe convergent light. In FIG. 3, only one probe is shown in each targetpolynucleotide hybridization area to be easy to read, but in practice, aplurality of probes having an identical base sequence are generallyimmobilized to each area.

According to the present embodiment, where the electrically conductivefilm 131 is placed on the surface of the substrate base 13, probesand/or polynucleotides can be elongated by providing a counter electrodeplate (not shown) above the substrate 1 and applying an alternatingfield between the electrode plate and the substrate, as described above.By this configuration, erroneous hybridization in the hybridization stepand steric entanglement in the denaturation of the target polynucleotidefrom the probe by heating the target polynucleotide hybridization areacan be prevented to ensure an efficient processing.

In addition, the cooling effect through the absence of the convergentlight 51 irradiation can be improved by using the thermally conductiveinsulating substrate 132 as the substrate base 13.

The photoabsorbing layer 21 according to the present embodiment may beprepared by vapor deposition, as well as by coating or spraying. Thetarget polynucleotide hybridization areas can be formed in the shape ofsquare or rectangular, as well as round or ellipse in accordance withthe shape of the convergent light.

FIG. 4 illustrates a second means for heating a specific area on thesubstrate 1. The photoabsorbable thin layer 21 is formed on the targetpolynucleotide hybridization areas in the embodiment of FIG. 3, whereas,in the present embodiment, particles 23 each having photoabsorbingcharacteristics and have sufficiently small sizes in comparison withthose of the target polynucleotide hybridization areas are dispersed andplaced on the target polynucleotide hybridization areas. At least oneparticle should be placed on each area. According to the presentembodiment, heat insulating layer 22 is separately provided in each ofindividual areas and the particles 23 are placed onto the upper surfaceof the insulating layer 22. The substrate 1 comprises substrate base 13composed of electrically conductive film 131 and thermally conductiveinsulating substrate 132 as well as in the embodiment illustrated inFIG. 3.

When the convergent light 51 is irradiated to a specific targetpolynucleotide hybridization area 142, the particle 23 in the areaabsorbs the light to evolve heat and hence the vicinity of the area 142alone is increased in temperature so that the target polynucleotidealone being hybridized to the probe on the area 142 can be denaturedfrom the probe. According to the present embodiment, the use ofparticles 23 ensures an area, smaller than the convergent range of theconvergent light of the present embodiment of FIG. 3, to be heatedspecifically, whereas the size of each target polynucleotidehybridization area and that of the convergent area of the convergentlight have a similar relation as in the embodiment of FIG. 3. Thephotoabsorbing layer composed of the particles 23 according to thepresent embodiment can be prepared by coating or spraying of particles.The particles can be shaped arbitrarily as in the embodiment of FIG. 3.

FIG. 5 illustrates a third means for heating a specific area on thesubstrate 1. The present embodiment is similar to the embodiment of FIG.4 in that temperature increase by particle 24, which absorbs light atwavelength region of the convergent light, is utilized, but differentfrom the latter in that the particle 24 is captured by the convergentlight 51 in the manner of optical forceps, and the captured particle 24is placed in a target polynucleotide hybridization area on which probeand the target polynucleotide are to be denatured. The numericalaperture of the objective lens 15 in this case should preferably beequal to or more than 1.2 for ensuring satisfactory characteristics asoptical forceps. Heat insulating layer 22 is also provided respectivelyin each area in the present embodiment. Substrate 1 comprises substratebase 13 composed of electrically conductive film 131 and thermallyconductive insulating substrate 132 as well as in the embodiment of FIG.3. As the embodiment of FIG. 5 is similar to the embodiment of FIG. 4except that the particle 24 is captured in the manner of opticalforceps, other descriptions concerning the figure are omitted.

According to the present embodiment, temperature increase by theparticle 24 is confined within the vicinity of the particle 24 in thearea where the particle 24 is placed, the sizes of the targetpolynucleotide hybridization areas and that of the convergent area ofthe convergent light have no fundamental relationship. To be morespecific, temperature increase by the particle 24 is limited to thevicinity of the particle 24 in the area where the particle 24 is placed,and hence a target polynucleotide to be denatured is limited to thatpresent in the target polynucleotide hybridization area where theparticle 24 is place, even when the convergent area of the convergentlight covers a plurality of target polynucleotide hybridization areas.The present embodiment is, therefore, advantageous in that the targetpolynucleotide hybridization areas can be arranged with an increaseddensity.

FIG. 6 demonstrates a more detailed illustrative configuration includingthe relationship between the nucleotide separation cell 7 and thesubstrate 1 using the basic configuration of the embodiment shown inFIG. 2.

Part of the polynucleotide separation cell 7 in FIG. 6 is notched forviewing the inside thereof. FIGS. 7 and 8 are sectional views of thepolynucleotide separation cell 7 respectively along with the lines A—Aand the lines B—B.

The polynucleotide separation cell 7 is provided with upper cell plate721, lower cell plate 722, and three sample solution chambers 731, 732and 733 which are partitioned by a plurality of spacers 723. The uppercell plate 721 is fabricated of a light transmittable material. The treesample solution chambers can import and export a sample solution throughsample solution inlets 711, 712 and 713 respectively. Between each ofthe sample solution chambers are connected communication holes 714 and715, through which the sample solution is transferred. The substrate 1on which probes are immobilized as illustrated in FIG. 2 is mounted onthe lower cell plate 722 in the sample solution chamber 732, and on thesubstrate 1 is integrated electrode plate 131 for applying an electricfield. Electrodes 751, 752, 754 and 755 are attached to individual sidewalls of the sample solution chambers 731, 732 and 733 respectively, andmesh electrode 753 is placed on the inner wall of the upper cell platefor applying an electric field onto above the sample solution chamber732. The temperature of the substrate 1 can be controlled within therange from 0° C. to 95° C. through Peltier devices 32, 34 and 35 eachprovided with a cooling plate 33, and individual temperatures of thesample solution chambers 731, 732 and 733 can be independentlycontrolled within the range from 0° C. to 95° C. by operating thePeltier devices 32, 34 and 35 independently. Accordingly, the PCRreaction or denaturation reaction can be carried out in each of thesample solution chambers without changing the temperatures of the otherchambers. To move the position of the substrate 1 to be observed throughthe objective lens 15, the cell 7 is fixed on rails 742, 743 and 744 byjig 741 and can be arbitrarily transferred on a two-dimensional plane bya stepping motor (not shown).

FIGS. 9A through 9C illustrate timetables demonstrating a polynucleotideseparation process using the polynucleotide separation cell shown inFIGS. 6-8. Initially, a sample solution containing polynucleotidesintroduced into the sample solution chamber 731 is subjected to PCRamplification as a pretreatment by applying a total of about 20 to 30cycles of chronological changes in temperature to the solution as shownin FIG. 9A. In other words, double stranded-polynucleotides aredenatured into single stranded-polynucleotides in the first denaturationprocess. Annealing with probes in the sample solution and subsequentlypolymerase elongation are then conducted, and these processes arerepeated to amplify polynucleotides. The temperatures in the processesare controlled on the whole sample solution in the sample solutionchamber 731 by the Peltier device 32. From the sample solutioncontaining the amplified polynucleotides, a polynucleotide having aspecific base sequence is extracted by means of process as shown in FIG.9B. To be more specific, in the sample introduction process,polynucleotides in the sample solution in the sample solution chamber731 are induced to the sample solution chamber 732 by setting theelectrodes 751 and 755 as cathodes and the electrode 754 and theelectrode above the substrate 1 as anodes. The sample solution in thesample solution chamber 732 is then heated to 95° C. by the Peltierdevice 34 to disassociate hydrogen bonds in polynucleotides in thesample solution to form single stranded-polynucleotides. The singlestranded-polynucleotides are then hybridized to probes in the targetpolynucleotide hybridization area on the substrate 1 by cooling thesolution to a temperature of 37° C. Remained polynucleotides in thesample solution not hybridized to the probes in the hybridization areasare then returned to the sample solution chamber 731 by setting theelectrode 751 as an anode. The cell is then subjected to fluorescenceobservation to identify to which area on the substrate 1 the targetoligonucleotide is hybridized. On the basis of information of an areawhere the target polynucleotide is hybridized to the probes on thesubstrate 1 (output of the fluorescent emission intensity detector 191shown in FIG. 2), the target polynucleotide hybridized to the probe in atarget polynucleotide hybridization area on the substrate 1 can bedissociated from the probe by irradiating a convergent light to thearea. In this process, an alternating field is applied between theelectrode 753 and the electrode 131 on the surface of the substrate 1 toelongate the target polynucleotide. In addition, the targetpolynucleotide in the specific target polynucleotide hybridization areaon the substrate 1 alone is denatured by supplying the convergent light51 from the lens 15, while setting the electrode 755 as an anode, andthus the objective target polynucleotide alone is introduced into thesample solution chamber 733. By setting the electrode 752 as a cathodewhile retaining the electrode 751 as an anode, polynucleotides in thesample solution in the sample solution chamber 731 which have not beenhybridized to the probes are retained in the sample solution chamber 731without transferring to the sample solution chamber 732 or 733.Components required for PCR are then introduced through the solutioninlet 713 to the sample solution chamber 733 while continuously applyingan electric field to the electrodes. About 20 to 30 cycles ofaftertreatment shown in FIG. 9C are then carried out to extract andamplify an objective polynucleotide selectively with a high precisionand a high speed from the sample solution introduced into the samplesolution chamber 733. After extracting the amplified sample solution inthe sample solution chamber 733, each of polynucleotides hybridized toeach of the areas can be selectively extracted and amplified in sequenceby moving the substrate 1 and repeating the separation process shown inFIG. 9B and the aftertreatment PCR process shown in FIG. 9C in a similarmanner as above.

Specific procedures for decreasing the temperatures are not described inthe timetables shown in FIGS. 9A through 9C, whereas cooling ofindividual chambers can be conducted in a short time by positively usingthe Peltier devices 32, 34 and 35 in FIG. 6 for heat dissipation. SuchPeltier devices can be provided separately in individual targetpolynucleotide hybridization areas for cooling after the area specificdenaturation for denaturing polynucleotides hybridized to the probes ofthe target polynucleotide hybridization area.

Embodiment II

Embodiment II is essentially identical with Embodiment I except that theformer utilizes heating elements embedded in the substrate 1 fortemperature control of individual target polynucleotide hybridizationareas to denature the target polynucleotide hybridized to the probes,instead of the convergent light irradiation in the latter. The presentembodiment will now be described in detail with reference to FIGS. 10through 15.

FIG. 10 illustrates the relationship between substrate 1 having a targetpolynucleotide hybridization area and an optical system for detectinghybridization in the target polynucleotide hybridization area, accordingto the second embodiment of the invention. Comparison between FIG. 10and FIGS. 2 and 3 demonstrates that the substrate according to thepresent embodiment comprises substrate base 13 composed of electricallyconductive film 131 having target polynucleotide hybridization areas onits surface, temperature control unit 133 and thermally conductiveinsulting substrate 132. On the surface of the electrically conductivefilm 131 are provided a plurality of target polynucleotide hybridizationareas 141, 142, 143, 144, 145 and 146, and inside of the temperaturecontrol unit 133 are embedded electrodes and heating elements to evolveheat by the electrodes, for independently controlling the temperaturesof individual target polynucleotide hybridization areas separately. Eachof the target polynucleotide hybridization areas has a dual-layerstructure composed of probe hybridization layer 221 such as an aluminumoxide thin film layer, and electric insulator layer 222. Probes(oligonucleotides) 41, 42, 43, 44, 45 and 46 each having a length of 8-9bp are respectively immobilized to the surface of each of the targetpolynucleotide hybridization areas. FIG. 10 schematically illustratesthe state where complementary target polynucleotides 41′, 42′, 43′ and46′ alone are respectively hybridized only to the probes 41, 42, 43 and46 of these probes.

When the present embodiment is configured such that a fluorescent dye isattached simultaneously at the time when the target polynucleotides 41′,42′, 43′ and 46′ are hybridized to probes 41, 42, 43 and 46, themagnitude of hybridization between the probe and target polynucleotidein each area can be estimated through fluorescence emission intensity byexciting the fluorescent dye and detecting fluorescence emitted from thedye. In addition, area specific temperature increase in the targetpolynucleotide hybridization areas can be determined by using thephenomena where the fluorescence emission intensity of a fluorescent dyedecreases in proportion to an increasing temperature through thefunction of the thermal quenching effect. The temperatures of targetpolynucleotide hybridization areas can be estimated by analyzing thefluorescence emission intensity changes of the fluorescent dye basedupon its temperature dependency. The identical or equivalent elements tothose of EMBODIMENT I are indicated with the same reference numerals inthe present embodiment.

FIG. 11 is a sectional view of the substrate 1 shown in FIG. 10 alongwith the lines A—A. Substrate 1 has a laminate structure composed ofthermally conductive insulating substrate 132, temperature control unit133 and electrically conductive film 131. On the surface of the toplayer, electrically conductive film 131, is formed a dual-layerstructure of probe hybridization layer 221 to be target polynucleotidehybridization area, and electrically insulating layer 222. Below thetemperature control unit 133 are formed layers of planar electrodes 226,heating elements 225 and planar electrodes 224. Individual electrodes inthe planar electrode 226 and the planar electrode 224 are in the shapeof cross-matrix such that individual crossing points separatelycorrespond to the position of individual heating elements and topositions of individual target polynucleotide hybridization areas.Thermistors 231 for temperature measurement are embedded in thethermally conductive insulating substrate 132 corresponding to each ofthe target polynucleotide hybridization areas, by which the temperatureof each of the hybridization areas can be determined.

FIGS. 12A through 12C are sectional views of the temperature controlunit 133 of the substrate 1 according to the second embodiment alongwith the lines B—B, lines C—C and lines D—D, respectively. The planarelectrodes 226 are orthogonal to the planar electrodes 224, andindividual crossing points therebetween sandwich each of the heatingelement layers 225. Consequently, a heating element layer 225 alone at acrossing point of a planar electrode 226 and a planar electrode 224 canevolve heat by selectively applying a potential difference between theabove planar electrode 226 and the above planer electrode 224 to pass anelectric current. In other words, by selecting a planar electrode 226and a planar electrode 224 properly and applying a potential differencetherebetween, the temperature of a specific target polynucleotidehybridization area corresponding to the crossing point between theseselected electrodes can be increased.

The substrate 1 shown in FIG. 10 can be used in place of the substrate 1of the polynucleotide separation cell 7 shown in FIGS. 6 through 8,whereas a simpler embodiment will be described herein.

FIG. 13 is a block diagram illustrating a polynucleotide separation cell251 and related devices thereto, and FIG. 14 is a sectional view of thepolynucleotide separation cell 251 along with the lines G—G, eachaccording to the second embodiment. The polynucleotide separation cell251 is composed of a is light-transmittable case to permit opticalobservation of the substrate 1 in the cell. To the cell 251 areconnected sample solution injection tube 252 for injecting a samplesolution containing polynucleotides, and extraction tube 253 forextracting the target polynucleotide hybridized to a specific targetpolynucleotide hybridization area on the substrate 1, through which thesolution can be passed in the direction indicated by arrows 57 and 58.Above the cell 251 is provided transparent electrode (not shown) forapplying a potential difference between the transparent electrode andthe electrically conductive film 131 of the substrate 1. The presentembodiment includes direct current power source 254, alternating currentpower source 255 and control circuit 256 for allowing a heating elementcorresponding to a specific target polynucleotide hybridization area toevolve heat or for generating a DC field or alternating field in thetarget polynucleotide hybridization area. The state of the substrate 1can, therefore, be controlled on the basis of temperature informationfrom the thermistors 231 according to procedures mentioned below.

FIG. 15 is a timetable illustrating a separation process in thepolynucleotide separation cell according to the second embodiment.

From a sample introduced into the cell 251, a polynucleotide having aspecific base sequence is singly extracted in the process shown in FIG.15.

(1) In the sample solution introduction process, polynucleotides in thesolution are induced to the surface of the substrate 1 by setting theelectrically conductive film 131 of the substrate 1 as an anode and thetransparent electrode on the upper surface of the cell as a cathode.Separately, polynucleotides can be elongated by generating analternating field between the electrically conductive film 131 of thesubstrate 1 and the transparent electrode on the upper surface of thecell. In this step, the surface temperature of the substrate 1 isincreased up to 95° C. by allowing all electrodes 224 and 226 to beconnected and to thereby all heating elements 225 to evolve heat, eachin the temperature control unit 133.

(2) The temperature is then decreased to 37° C. This procedure allowsthe probes immobilized on the target polynucleotide hybridization areasto hybridize to the target polynucleotides in the sample solution.

(3) Next, the surface layer of the substrate 1 is heated up to about 72°C., and the electrically conductive film 131 of the substrate 1 and thetransparent electrode on the upper surface of the cell are respectivelyset to a cathode and an anode so as to remove, together with the samplesolution, polynucleotides non-specifically hybridized to probes.

(4) After replacing the solution in the cell 251 with a new one, eachone of the electrodes 224 and 226 is respectively allowed to beconnected, and one of the heating elements 225 is allowed to evolveheat, each in the temperature control unit 133 to increase thetemperature of one of the target polynucleotide hybridization areas upto 95° C., thereby the target polynucleotide hybridized to probes inthis area is denatured and extracted with the solution.

(5) By repeating similar procedures on each of the target polynucleotidehybridization areas, each of target polynucleotides hybridized to probesin individual areas can be extracted in sequence.

In this case, the processing efficiency can be enhanced by detecting,beforehand, hybridization state of target polynucleotides on individualtarget polynucleotide hybridization areas, on the basis of temperatureinformation from the detector 191 and the analyzer 192 and conductingthe temperature control only on an area to which a polynucleotide to beextracted is hybridized, as described in EMBODIMENT I.

Embodiment III

EMBODIMENT III relates to a configuration of substrate 1 having targetpolynucleotide hybridization areas which are designed to immobilizeprobes with stability. To be more specific, the present embodimentproposes the substrate 1 having a configuration in which the surfaces ofhybridization areas are fabricated of oxidized metal films and theprobes can be immobilized with stability by a silane coupling reaction.

The substrate 1 according to the present embodiment may be prepared inthe following manner: A 0.4-mm thick and 24-mm square glass substrate isimmersed in an NaOH is (1 M) solution and subjected to ultrasoniccleaning for 30 minutes. The cleaned substrate is washed with runningultrapure water and thereafter baked at 110° C. for 15 minutes. Using avacuum metallizer, chromium (Cr) is vacuum-deposited on the substrate ina thickness of 3 nm, and the deposited substrate is washed with ethanol.After immersed in 3-glycidoxypropylmethoxysilane (not diluted) for 5minutes, the substrate is immersed in a 4%3-glycidoxypropylmethoxysilane solution in 50% ethanol medium for 30minutes with stirring at times. The substrate is then taken out from thesolution and baked at 110° C. for 30 minutes to introduce glycidoxygroups onto the surface of the metal through the silane couplingreagent, and thereby substrate 1 is obtained.

FIG. 16 illustrates the stable immobilization of probes on a substrate 1according to EMBODIMENT III. The substrate 1 according to the presentembodiment is composed of glass substrate 302, Cr layers 303 depositedon the substrate 302, which surfaces constitute oxidized films 304.Probes are immobilized through silane coupling layers 305. To verifythat the probes are immobilized with stability according to the presentembodiment, polynucleotides respectively having lengths of 521 bp and625 bp, whose ends are labeled with fluorescent dye sulforhodamine 101,are hybridized to probes 311 and 312, which probes are respectivelyimmobilized through silane coupling layer 305 on target polynucleotidehybridization areas 306 and 307.

A comparative test to verify the advantages of the substrate 1 accordingto EMBODIMENT III will be described below.

Initially, comparison was conducted among the substrate according toEMBODIMENT III, a substrate obtained by vacuum-depositing aluminium (Al)instead of Cr on the surface of glass 302, and a substrate obtained bysubjecting the surface of glass 302 directly to silane coupling. As thesubstrate according to the present embodiment, two substrates wereprepared by vacuum-depositing Cr on the whole surface of glass 302 in athickness of 5.4 nm. These substrates had light transmittance at 600 nmto 800 nm of 53% to 56%. The Al-deposited substrate was obtained byvacuum-depositing Al instead of Cr on the whole surface of glass 302 ina thickness of 15 nm in a similar manner to that in the embodiment. Thesubstrate had a light transmittance at 600 nm to 800 nm of 19 to 25%.Glass itself without deposition of a metal film has a lighttransmittance at 600 nm to 800 nm of about 84%. Within these ranges oftransmittance as above, a target polynucleotide hybridized to a probecan be detected through fluorescence emitted by exciting the targetpolynucleotide labeled with a fluorescent dye such as the sulforhodamine101 (absorption maximum 594 nm, fluorescence 615 nm) by exciting lightderived from argon (Ar, 514 nm) or helium-neon (He—Ne, 545 nm).

FIGS. 17A through 17C are diagrams illustrating results of tests toverify the advantages of the substrate according to EMBODIMENT III.

Using a series of tested substrates, polynucleotides respectively havinglengths of 521 bp and 625 bp, whose ends were labeled with fluorescentdye sulforhodamine 101 were hybridized to probes 311 and 312, whichprobes had been respectively immobilized through silane coupling layer305 on each of the tested substrate. The treated substrates weresubjected to laser confocal microscopic scanning, and the resultantrelative fluorescence emission intensity and transmittance are shown inFIG. 17A for a substrate according to EMBODIMENT III obtained byvacuum-depositing Cr 5.4 nm thick, FIG. 17B for a substrate obtained byvacuum-depositing Al 15 nm thick instead of Cr, and FIG. 17C for asubstrate having no deposited metal film. In the figures, the abscessesindicate the scanning position on the substrate, and the vertical axesindicate, by solid line 330, fluorescence emission intensity (equal toor more than 580 nm) distribution and transmitted light intensitydistribution detected with the laser microscope; and the broken line 331indicates transmittance distribution at wavelengths from 380 nm to 450nm.

FIG. 17A demonstrates that a constant fluorescence emission intensity330 could be obtained all over the area CA where the polynucleotidelabeled with the fluorescent dye sulforhodamine 101 was hybridized, inthe substrate according to EMBODIMENT III. The fluorescence emissionintensity 330 in the other positions was sufficiently low. Thetransmittance at 380 nm to 450 nm was almost constant in any positions,indicating that the Cr layer was retained with stability and was notdelaminated from the surface of glass 302 over the reactions andprocedures from immobilization of probes to hybridization of the targetpolynucleotide.

FIG. 17B for the Al-deposited substrate instead of Cr depositiondemonstrates that, the transmittance of the area CA where thepolynucleotide labeled with the fluorescent dye sulforhodamine 101 wasto be hybridized was remarkably high, indicating that the Al-depositedsurface had been dissolved in the immobilization of the probes. In otherwords, the Al-deposited surface was dissolved in a 10 mMTris-hydrochloric acid-1 mM EDTA buffer solution (pH 7.5) which is agenerally used medium for dissolving DNA and/or RNA, indicating thatsuch an Al-deposited substrate cannot be used in reaction systems usingaqueous solutions. Consequently, in this tested substrate, the probeswere not substantially immobilized, hence the polynucleotide labeledwith the fluorescent dye sulforhodamine 101 was not hybridized, and thefluorescence emission intensity was significantly low globally.

As is shown in FIG. 17C for the substrate where the probes wereimmobilized by subjecting the surface of the glass directly to silanecoupling, the area CA where the polynucleotide labeled with thefluorescent dye sulforhodamine 101 was hybridized had a higherfluorescence emission intensity 330 than the other positions where noprobes were immobilized, indicating that the substrate served toimmobilize the probes. The fluorescence emission intensity 330 in thissubstrate fluctuated, however, by location as compared with thesubstrate according to the present embodiment, indicating that theprobes were not immobilized homogeneously.

The above results demonstrates that the Cr-deposited substrate accordingto the present embodiment is advantageous for stable immobilization ofprobes and for hybridization of target polynucleotides. In addition toCr-deposited substrates, any substrates each obtained by vacuumdepositing a metal, which is stable in a weak alkali solution, on aglass substrate in a thickness of several nanometers to 10 nm areadvantageous. To be more specific, those covered with a film of oxide onthe surfaces and hardly dissolved in a weak alkali solution at 95° C.are desirable, whereas gold and platinum cannot be used since they formno oxide film and hence silane coupling cannot be done. Stainless steelhas similar characteristics to Cr such as to be stable in a weak alkalisolution and to form an oxide film, but it is excluded from the scope,since it does not allow active residues to be introduced by silanecoupling for some unknown reasons. Similar examinations have revealedthat at least any metal of Ti, V, Cr, Fe, Co, Ni, Mo and W is effectiveand advantageous.

Next, explanation will follow concerning that a target polynucleotidehybridized to a probe on the substrate according to the presentembodiment can be efficiently extracted, with reference to FIGS. 18A and18B. FIGS. 18A and 18B respectively illustrate the results of testswhere polynucleotides respectively having lengths of 521 bp and 625 bp,whose ends were labeled with fluorescent dye sulforhodamine 101, werehybridized to probes 311 and 312, which probes were respectivelyimmobilized through silane coupling layer 305 on the areas 306 and 307shown in FIG. 16. The surface of each tested substrate was covered with20 microlitter of a 20 mM Tris-hydrochloric acid buffer (pH 7.5).

FIGS. 18A and 18B respectively demonstrate the results obtained by laserscanning (514 nm) over the areas 306 and 307 separately and detectingfluorescence emission intensity (510 nm to 580 nm), with the abscissaindicating scanning positions on the substrate and the vertical axisindicating relative fluorescence emission intensity distributiondetected through a laser microscope. Line 335 represents the relativefluorescence emission intensity, indicating that a nearly constantfluorescence was detected all over the both areas 306 and 307. Next, thearea 306 alone was subjected to scanning with 10 mW YAG laser (spotdiameter 5 nm) at 1053 nm. As shown in FIG. 18A, the fluorescenceemission intensity after scanning was decreased to about one-tenth atthe region 333 which scanned with YAG laser, as indicated by broken line336. In the area 307, both fluorescence emission intensities 335 and 336did not change.

FIG. 19 schematically illustrates results of electrophoresis concerningfractionation of polynucleotides according to the present embodiment.

Lane 340 is a lane of a marker. Lanes 341 and 342 demonstrate theresults of electrophoresis on identical polynucleotides with thoseobtained in the areas 306 and 307 of the substrate 1 respectively. Bandsof 521 bp and 625 bp are clearly observed in the lanes 341 and 342,respectively. Lanes 343 and 344 demonstrate the results obtained byscanning the area 306 of the substrate 1 with 10-mW YAG laser (spotdiameter 5 nm) at 1053 nm, recovering the buffer on the surface of thescanned substrate 1, subjecting the buffer for PCR amplification, andsubjecting the PCR amplification products to 2% agarose gelelectrophoresis analysis. A band of 521 bp is clearly detected in thelane 343, whereas no band of 625 bp is detected in the lane 344,indicating that only the polynucleotide denatured from the area 306,which has been scanned with 10-mW YAG laser (spot diameter 5 nm), ispresent in a solution obtained by subjecting the recovered buffer to PCRamplification. According to the present embodiment, immobilization ofprobes and an efficient fractionation of hybridized polynucleotides canbe achieved.

Embodiment IV

EMBODIMENT IV is designed to utilize the inside wall of a capillary forminimizing the amount of a sample solution required for fractionation ofpolynucleotides, whereas the substrate 1 each in the above EMBODIMENTS Ithrough III is plane-shaped.

FIGS. 20 and 21 illustrate an external view and a sectional view alongwith the lines A—A of a capillary applicable in the fourth embodiment.

Capillary 401 is a light transmittable capillary such as a glasscapillary. Inside of capillary wall 412 is coated with inner coat 413composed of Cr or other metal exemplified in EMBODIMENT III. The surfaceof the inner coat is composed of a stable oxide formed by air oxidationof the metal. On the surface of the coat are cylindrically placed targetpolynucleotide hybridization areas 414, 415 . . . on which probes areimmobilized. Area specific probes are closely immobilized on each of thetarget polynucleotide hybridization areas 414, 415 . . . . Ofpolynucleotides in a sample solution, only each of polynucleotidescomplementary to each of the probes can be hybridized to each of theareas. On the external surface of the capillary 401 are indicatedmarkers 411 at the boundary between the individual areas so as to ensurethe observation of the positions of individual target polynucleotidehybridization areas from the outside of the capillary.

FIG. 22 is a diagram of an illustrative configuration for immobilizing aprobe on a target polynucleotide hybridization area.

Independent oligonucleotide probe layers can be formed concentrically inthe inner wall of a capillary, as illustrated in FIG. 21, for example,in the following manner: Initially, epoxy groups 416 are formed all overareas on the inner wall of the capillary 401 by reacting the whole innerwall of the capillary with a divalent reagent having silanol group andepoxy group at both ends to make the inner wall water repellent andthereby reject an aqueous sample solution. Next, a nucleotide samplesolution to be hybridized to a specific area 415 is introduced as a dropinto the capillary 401. The drop becomes spherical and in concentriccontact with the inner wall with a minimum contact area because of thewater repellency of the inner wall of the capillary. The drop is thenintroduced to the area 415 and left for a while to make a free end ofthe epoxy group 416 to hybridize to an oligonucleotide probe, which5-end has been modified with an amino group. The drop containing thereacted nucleotide probe is taken out of the capillary 401 immediatelyafter the completion of the reaction. By repeating procedures in which adifferent sample drop is introduced to react in another area in asimilar manner, the configuration shown in FIG. 21 can be obtained.

The timetable for fractionating target polynucleotides in a samplesolution according to the fourth embodiment is not described herein asit is identical with the timetable shown in FIG. 15.

FIG. 23 is a general view of the configuration of a polynucleotideseparation apparatus according to the fourth embodiment. In the insideof polynucleotide separation module 431 is integrated the capillary 401described in FIGS. 20 and 21. Both ends of the capillary 401 areseparately connected to capillary connection units 432 and 433. One of asample solution retained in reservoir 434, a washing solution retainedin reservoir 435, a washing solution retained in reservoir 436 and airpassing through air filter 437 is selected by selector 438, andintroduced into the capillary connection unit 432 with pump 439.Controller 400 represents a control and power supply means according tothe apparatus of the present embodiment. The solution extracted from thepolynucleotide separation module 431 passes through the capillaryconnection unit 433 and is supplied to aftertreatment process 431including PCR amplification. To the controller 400 are collectednecessary data from individual units of the apparatus, and necessaryoperation signals in accordance with the data are sent to individualunits from the controller. Details of a practical configuration andconnection to individual units of the apparatus are omitted herein.These can be achieved by one skilled in the art based upon theaforementioned timetables and the following descriptions.

FIG. 24 is a sectional view along with the lines B—B of thepolynucleotide separation module 431 of the fourth embodiment. In theinside of the module 431 are provided, in parallel, light source 442 forintroducing a light to the wall of the capillary 401; doughnut-shapedheat sources 443 corresponding to individual target polynucleotidehybridization areas of the capillary 401; light detection probes 444 fordetecting fluorescence emitted from a target polynucleotidehybridization area which is excited by an exciting light plasmon-excitedfrom the capillary to the inside, or for detecting the markers 411 onthe surface of the capillary; and thermal detection probes 445 fordetecting the temperature of the capillary. Electrodes 451 and 452 areprovided respectively in the capillary connection units 432 and 433 toextract sample DNA in the capillary 401 by electrophoresis. Electrodeconnector 453 is to apply an electric potential to the inner coat 413 ofthe capillary 401 so as to move nucleotides toward or away from theinner surface of the capillary.

FIG. 25 illiterates a variation of the polynucleotide separation module431 shown in FIG. 24 according to the fourth embodiment. The basicconfiguration of the variation is the same as in the embodiment shown inFIG. 24, except that the former employs drop 471 as a washing solutionto be introduced for extracting a separated polynucleotide. The drop 471is introduced from the reservoir 436 into the capillary 401 by adjustingthe selector 438 shown in FIG. 23; the drop 471 is then pushed to anobjective target polynucleotide hybridization area by air passingthrough the air filter 437 so as to cover the area. Next, leak valves461 and 462 are opened to escape air in the capillary 401, which isexpanded by area specific heat from the heat source 443, the position ofthe drop 471 is thus fixed. The temperature of the area is thenincreased to a temperature equal to or higher than the denaturationtemperature to denature the target polynucleotide from the probe.Subsequently, the leak valves 461 and 462 are closed, and the drop 471is pushed to the capillary connection unit 433 by the air passingthrough the air filter 437 so as to extract the target polynucleotide.According to the present embodiment, the drop come in contact with aspecific target polynucleotide hybridization area alone, andcontamination to the drop will not occur even if the temperature ofother area reaches to the denaturation temperature. The extraction witha higher precision can, therefore, be achieved. In the presentembodiment, area specific heating is carried out by one of the heatsources 443 in the polynucleotide separation module 431, whereas theextraction with a high precision can also be achieved by using one heatsource in the shape covering all over the target polynucleotidehybridization areas.

FIG. 26 illustrates another variation of the polynucleotide separationmodule 431 according to the embodiment shown in FIG. 24. In the presentembodiment, light source 442, heat source 443, light detection probe 444and heat detection probe 445 each to be provided in the inside of thecapillary 401 are omitted, and an optical system is provided in theexterior to the capillary 401 as an alternative to the above elements.In other words, the present embodiment employs the capillary 401 insteadof the substrate 1 in the configuration shown in FIGS. 2 and 6 accordingto EMBODIMENT I. Consequently, the identical optical system with that inFIG. 2 can be employed, whereas only the objective lens 15 is shown inFIG. 26 for the simplification.

The capillary 401 connected to the capillary connection units 432 and433 is moved on the rail 446 to observe the state of the inside of thecapillary at a specific target polynucleotide hybridization area; andsimultaneously a convergent light is irradiated to the aforementionedarea of the capillary to heat area specifically the vicinity alone ofthe convergent point in the capillary. When the capillary is designed torotate in the circumferential direction, the state of the hybridizationof polynucleotide can be observed, heated and extracted all over theareas in the circumferential direction in the capillary.

FIG. 27 illustrates a variation of the polynucleotide separation module431 according to the embodiment shown in FIG. 26. The present variationis similar to the embodiment in FIG. 26 in that an optical system isprovided in the exterior of the capillary 401 but different in that onlyone set of the optical system identical with those to be provided inindividual target polynucleotide hybridization areas in the capillary isprovided in the exterior of the capillary to thereby constitute module431, and that the module 431 is designed to be moved on rail 481 withrespect to the capillary 401. By configuring like this, the apparatuscan globally be miniaturized.

Embodiment V

The aforementioned EMBODIMENTS I through IV are applied to the casewhere a target polynucleotide is contained in a sample solution, whereasEMBODIMENT V is directed to fractionate a target polynucleotide directlyfrom cells themselves. According to the present embodiment, naturally,the target polynucleotide is finally fractionated in the form of asample solution containing the same. Such a sample solution is, however,obtained after introducing cells to a target polynucleotidehybridization area so that the amount of target polynucleotide in thesample solution can be increased, and hence PCR or other pretreatmentcan be omitted in many cases. In addition, nucleotides, proteins andother cell components can be obtained for each of the cells according tothe present embodiment.

FIG. 28 illustrates basic elements of the present embodiment.

On the surface of substrate 1 is formed target polynucleotidehybridization area 511 as in described in the above EMBODIMENTS.Controller 521 is to apply a DC or alternating field between anelectrode of the hybridization area 511 and grounding electrode 514opposed to the substrate 1 to capture and hybridize cell 561 in thehybridization area 511. Power supply 526 is for applying a DC oralternating field or grounding, including selection switch 525. Peltierdevices 512 and temperature sensors 513 are embedded in the substrate 1separately in the vicinity of individual target polynucleotidehybridization areas. Temperature measurement and control unit 523 is toinput a signal from the temperature sensor 513 and to control thesurface temperatures of the individual target polynucleotidehybridization areas independently through Peltier devices 512.Controller 521 gives a direction on which area is to be controlled intemperature.

FIG. 29 illustrates a basic configuration according to the presentembodiment of a combination of a polynucleotide separation cell havingthe basic elements illustrated in FIG. 28 and an optical system. FIG. 30is a sectional view along with the lines A—A of the separation cellillustrated in FIG. 29.

Polynucleotide separation cell 541 is provided with sample inlet tube542 and sample outlet tube 543, through which sample cells, washingsolutions or the like are introduced into the separation cell, and cellresidues, proteins, purified nucleotide samples or the like arerecovered from the separation cell. In the inside of the separation cell541 are formed, as shown in FIG. 30, target polynucleotide hybridizationareas and are placed electrodes 551, 552, 553 . . . for introducing asample cell introduced from the sample inlet tube 542 to the targetpolynucleotide hybridization areas, and lines are connected to theelectrodes. In this figure, upper electrode 514 is not illustrated. Asingle stranded-oligonucleotide probe complementary to each targetpolynucleotide is individually immobilized in each target polynucleotidehybridization area.

The inside of the polynucleotide separation cell 541 can be observedthrough the same optical system as described in FIG. 2. To be morespecific, the substrate 1 is placed in the direction perpendicular tothe light axis of the objective lens 15, and an exciting light fromlight source 16 for fluorescent observation is induced via the bandpassfilter 151 and the dichroic mirror 181 to the objective lens 15. Theexciting light induced through the objective lens 15 excites afluorescent dye, which is attached accompanied with a targetpolynucleotide hybridized onto the substrate 1, and the excitedfluorescent dye emits fluorescence in an intensity in proportion to theamount of the target polynucleotide hybridized to probes on each area ofthe substrate. The emitted fluorescence in an intensity in proportion tothe amount of the target polynucleotide is collected through theobjective lens 15. Of the collected fluorescence, only a fluorescencehaving the wavelength to be detected is introduced via the emissionfilter 152 to the detector 191 to allow the observation of the inside ofthe separation cell 541.

In general, part of cell membrane of a cell is destroyed to liberatenucleotides, proteins and other components by heating. Consequently, byintroducing a sample solution containing cells into the polynucleotideseparation cell 541 and thereafter increasing the temperature of aspecific target polynucleotide hybridization area on the substrate 1 upto 95° C., a cell in the area is destroyed to liberate its components,and double stranded-polynucleotides can be denatured into singlestranded-polynucleotides. Subsequently, by decreasing the temperature ofthe hybridization area to 37° C., a single stranded-polynucleotidecomplementary to the probe in the area can be hybridized to the probe,among these target polynucleotides. The temperature of a specifichybridization area can be controlled by selecting a Peltier device 512corresponding to the area and passing an electric current therethrough.

A process will be described for fractionating a cell component andtarget polynucleotide directly from blood cells according to the presentembodiment, with reference to FIGS. 31A through 31E and FIGS. 32Athrough 32D.

Initially, the ion strength of a blood sample is decreased to destroyred blood cells, and thus a blood sample containing white blood cellsalone as cell components is prepared. FIG. 31A illustrates the statewhere the target polynucleotide hybridization areas 551, 552, 553 . . .are formed on the surface of the substrate 1. FIG. 31B illustrates thestate where the blood sample is introduced into the polynucleotideseparation cell 541 and white blood cells 561 float over the surface ofthe substrate 1. In this state, an alternating field is applied betweenthe electrodes of the target polynucleotide hybridization areas 551,552, 553 . . . and the electrode 514 so as to attract the white bloodcells 561 to the target polynucleotide hybridization areas. The gradientof electric field density generated between the electrode 514 and theelectrodes of individual hybridization areas is condensed at theindividual areas. An alternating field having a frequency of equal to ormore than 1 kHz and a density of equal to or more than 10 V/mm canpreferably used in this process. FIG. 31C illustrates the state wherewhite blood cells are induced to, and placed on the targetpolynucleotide hybridization areas. In this state, afluorescence-labeled antigen substance is introduced into the cell 541.FIG. 31D illustrates the state where only white blood cells capable ofmaking antibody response to the antigen substance (white blood cells 562and 563 hatched in the figure) are labeled and become to emitfluorescence. It is preferable in this state to cool the surface of thesubstrate to a temperature of about 4° C. and to ensure the amounts ofmRNA and the like in the cells not to change accompanied with thehybridization with the marker antigen. The hybridization capability ofthe antibodies on the surface of B cells or the like does not changewith a decreasing temperature. The position of a target polynucleotidehybridization area where a white blood cell emitting fluorescence can,therefore, be identified through the output of the detector 191. Thecomponent of a cell having reactivity with the antigen substance can beliberated into the sample solution by heating individual targetpolynucleotide hybridization areas in sequence to destroy the cells.FIG. 31E illustrates the state where only one white blood cell 563 ofthose hybridized on the individual target polynucleotide hybridizationareas is destroyed and disappears from the substrate 1. Accordingly, thecomponent of the specific white blood cell can be extracted byrecovering the sample solution in the polynucleotide separation cell 541while applying an alternating field to other cells in this state.

FIG. 32A illustrates the state where probes 671 are immobilized on onearea 616 of the target polynucleotide hybridization areas. FIG. 32Billustrates the state where white blood cell 662 is induced andhybridized to the hybridization area 616 in the above manner. After thewhite blood cell 662 is induced to the hybridization area 616 as thus,the sample solution is exchanged with a new one to decrease its pH toabout 4. This is because almost all of pK values of proteins range 4 orhigher and by decreasing pH to 4 or lower, total charges of proteinsbecome positive whereas the charges of polynucleotides become negative;and hence the both can be separated in a DC field. Under such a pHcondition, the temperature of the hybridization area 616 is increased bythe Peltier device 512 to destroy the white blood cell. Thereafter, bysetting the electrode in the target polynucleotide hybridization area616 as an anode and the electrode 514 on the opposite surface as acathode, polynucleotides 664 are collected to the hybridization area 616and proteins 663 are electrophoresed to the face of the counterelectrode 514. FIG. 32C is a schematic diagram illustrating the statewhere polynucleotides 664 and proteins 663 migrate by electrophoresis inthe opposite direction to each other. The proteins can easily beseparated from polynucleotides by replacing the solution in the cell. Inthis process, polynucleotides complementary to the probe 671 immobilizedon the hybridization area naturally hybridize to the probes.

Next, by setting the electrode of the hybridization area as a cathodeand the counter electrode 514 as an anode, the target polynucleotide 666hybridized to the probes 671 on the hybridization area 616 remains onthe area 616, while polynucleotides not hybridized are liberated intothe sample solution. Of polynucleotides in the white blood cell 662,those not hybridized to the probes 671 can be extracted as in a samplesolution by recovering the sample solution in the present step. FIG. 32Dschematically illustrates the state where polynucleotides 665 nothybridized to probes 671 migrate by electrophoresis.

Finally, a specific target polynucleotide hybridized to the probes in aspecific target polynucleotide hybridization area is liberated byincreasing the temperature of the specific area up to about 95° C. Byrecovering the sample solution in this step, the objected targetpolynucleotide such as mRNA can be extracted and recovered.

After completion of the aforementioned procedure for on white bloodcell, a next white cell is subjected to the same procedure to extract amRNA or other target polynucleotide per each white blood cell.Separately, the extraction can be conducted by dying cells withdifferent plural types of markers by a marker labeling technique andobserving them with different filters to select and extract a cellmeeting a plurality of conditions.

The total amount of the target polynucleotide can quantitativelydetermined through signals from the optical system in the state wherethe target polynucleotide is hybridized to the probes in the targetpolynucleotide hybridization area. Consequently, the present embodimentcan be applied to the determination of the total amount of mRNA in a Bcell or other white blood cell, and according to the embodiment, acondition can be clarified easily and rapidly by estimating the activityof a white blood cell having reactivity to a specific antigen.

The present embodiment can naturally be applied to arbitrary cells inaddition to white blood cells.

Other embodiments and variations will be obvious to those skilled inthis art, this invention is not to be limited except as set forth in thefollowing claims.

1. An apparatus for recovering a target polynucleotide in a cellcomprising: a substrate being disposed in a separation cell, wherein thesample solution containing cells each containing polynucleotides andprotein is supplied on a surface of the substrate, a plurality ofindependent areas are formed on the surface of the substrate and each ofa single-stranded oligonucleotide probes each having a specific basesequence is immobilized to each of the areas; capturing means forcapturing each of the cells one by one separately on each of the areas;means for applying a DC field onto a surface of one area of the areas;temperature measuring means for measuring a temperature of the surfaceof the substrate at one area of the areas; heating or cooling means forheating or cooling the surface of the substrate at the one area of theareas; and controling means for controling selectively the temperatureof the surface of the substrate at the one area on the basis of atemperature information obtained by the temperature measuring means, bycontrolling the heating or cooling means, wherein the controling meanscontrols the heating or cooling means so as to heat the surface of thesubstrate at the one area of the areas to a first predeterminedtemperature to destroy the cell captured at the one area, to liberatethe polynucleotides and the proteins from the cell captured at the onearea, and to denature the polynucleotides liberated from the cell so asto obtain single-stranded polynucleotides, and the controling meanscontrols the heating or cooling means so as to cool a solution whichcontains no polynucleotide and has a pH value of 4 or lower and withwhich the sample solution on the substrate is replaced, to a secondpredetermined temperature to form hybrids between the single-strandedpolynucleotides and the single-stranded oligonucleotide probes, so as tocapturing single-stranded target polynucleotides; wherein, afterseparating the single-stranded polynucleotides and the proteins, wherebythe hybrids remain on the one area, by electrophoresis under the DCfield applied onto the surface of the one area, based on a chargedifference between the single-stranded target polynucleotides and theproteins, in the solution having a value of pH being 4 or lower, byflowing a washing solution into the separation cell, whereby the cellsat the areas except for the one area remain on the areas and the hybridsremain on the one area, the washing solution is recovered to recover theproteins liberated from the cell; wherein, after separating thesingle-stranded polynucleotides not forming the hybrids, whereby thehybrids remain on the one area, by electrophoresis under the DC fieldapplied the surface of the one area, by flowing the washing solutioninto the separation cell, the washing solution is recovered to recoverthe single-stranded polynucleotides not forming the hybrid; wherein,after heating the surface of the substrate at the one area of the areasto denature the hybrids at the one area, so as to liberate thesingle-stranded target polynucleotides into solution, by flowing thewashing solution into the separation cell, the washing solution isrecovered to recover the single-stranded target polynucleotidesliberated from the cell; and wherein, by changing a position of the onearea of the areas, the washing solution is recovered to recover,separately, the proteins, the single-stranded polynucleotides notforming the hybrid, and the single-stranded target polynucleotides, foreach of the areas.
 2. An apparatus according to claim 1, wherein thecell is a white blood cell.
 3. An apparatus according to claim 1,wherein the single-stranded target polynucleotide is mRNA.
 4. Anapparatus for recovering a target polynucleotide in a cell comprising: asubstrate being disposed in a separation cell, wherein the samplesolution containing cells is supplied on a surface of the substrate, anda plurality of independent areas are formed on the surface of thesubstrate; capturing means for capturing each of the cells one by oneseparately on each of the areas; means for applying a DC field onto asurface of one area of the areas; temperature measuring means formeasuring a temperature of the surface of the substrate at one area ofthe areas; heating or cooling means for heating or cooling the surfaceof the substrate at the one area of the areas; controling means forcontroling selectively the temperature of the surface of the substrateat the one area on the basis of a temperature information obtained bythe temperature measuring means, by controling the heating or coolingmeans; and means for identifying the positions of the areas where thecells to be destroyed are present, wherein the controling means controlsthe heating or cooling means so as to heat the surface of the substrateat one of the identified positions to a first predetermined temperatureto destroy the cell captured at the surface of the one of the identifiedpositions, to liberate the polynucleotides and the proteins from thecell captured at the area of the one of the identified positions, and todenature the polynucleotide liberated from the cell so as to obtain asingle-stranded polynucleotide, and the controling means controls theheating or cooling means so as to cool a solution which contains nopolynucleotide and has a pH value of 4 or lower and with which thesample solution on the substrate is replaced, to a second predeterminedtemperature to form hybrids between the single-stranded polynucleotidesand the single-stranded oligonucleotide probes, so as to capturingsingle-stranded target polynucleotides; wherein, after separating thesingle-stranded polynucleotides and the proteins, whereby the hybridsremain on the area of the one of the identified positions, byelectrophoresis under the DC field applied onto the surface of the areaof the one of the identified positions, based on a charge differencebetween the single-stranded target polynucleotides and the proteins, inthe solution having a value of pH being 4 or lower, by flowing a washingsolution into the separation cell, whereby the cells at the areas exceptfor the area of the one of the identified positions remain on the areasand the hybrids remain on the area of the one of the identifiedpositions the washing solution is recovered to recover the proteinsliberated from the cell; wherein, after separating the single-strandedpolynucleotides not forming the hybrids, whereby the hybrids remain onthe area of the one of the identified positions, by electrophoresisunder the DC field applied onto the surface of the area of the one ofthe identified positions, by flowing the washing solution into theseparation cell, the washing solution is recovered to recover thesingle-stranded polynucleotides not forming the hybrid; wherein, afterheating the surface of the substrate at the one of the identifiedpositions to denature the hybrids at the area of the one of theidentified positions, so as to liberate the single-stranded targetpolynucleotides into solution, by flowing the washing solution into theseparation cell, the washing solution is recovered to recover thesingle-stranded target polynucleotides liberated from the cell; andwherein, by changing a position of the identified positions, the washingsolution is recovered to recover, separately, the proteins, thesingle-stranded polynucleotides not forming the hybrid, and thesingle-stranded target polynucleotide, for each of the identifiedpositions.
 5. An apparatus according to claim 4, wherein the cell is awhite blood cell.
 6. An apparatus according to claim 4, wherein thesingle-stranded target polynucleotide is mRNA.